We seek to develop an ultra-fast insulin analog formulation for the treatment of diabetes mellitus. Ultra-fast pharmacokinetic/dynamic (PK/PD) promises to enable superior performance of pump therapy (continuous subcutaneous insulin infusion; CSII) with enhanced safety and more robust integration with continuous glucose monitors (CGM). Our product promises to improve the safety and efficacy of algorithm-controlled closed-loop systems (the artificial pancreas); ultra-fast PK/PD would also facilitate post-prandial glycemic control in conventional multi-injection regimens. An innovative structural approach is proposed based on non-standard mutagenesis of the insulin molecule. This application has been revised based on the suggestions of the Study Section, most saliently to enhance its focus on stability and to provide supporting animal data. The current barrier to more rapid subcutaneous absorption is the rate of disassembly of the zinc insulin hexamer. This rate is controlled by the stability of an anti-parallel -sheet at the dimer interface of the hexamr. Partial progress toward accelerated disassembly following subcutaneous injection was achieved in the 1990s by conventional site-directed mutagenesis at the periphery of this -sheet. Insulin aspart (the active component of Novolog(R); Novo-Nordisk) and insulin lispro (the active component of Humalog(R); Eli Lilly) contain such partially destabilizing mutations (ProB28->Asp in Novolog; ProB28->Lys and LysB29->Pro in Humalog (KP)). Because their designs were limited by conventional mutagenesis, however, neither of these products could fully exploit structural strategies to promote disassembly of the core -sheet in the insulin hexamer. The core -sheet of the insulin hexamer is remarkable for the confluence of 8 conserved aromatic rings (TyrB16, PheB24, PheB25, TyrB26, and their dimer-related mates). The thermodynamic and kinetic stability of this interface is regulated by an intricate series of aromatic-aromatic interactions. We have discovered that the substitution of PheB24 by its non-aromatic analog cyclohexanylalanine (Cha) is compatible with native-like structure and function, but markedly enhances the rate of disassembly of KP-insulin. Since submission of the original application, we have also invented an ultra-stable version of this analog containing additional A-chain substitution ThrA8->Glu. We therefore propose to synthesize and characterize ChaB24-KP-insulin and ChaB24- GluA8-insulin as candidate ultra-rapid formulations. Dr. B. Frank (principal investigator) was co-inventor of Humalog(R) during his prior career at Eli Lilly. Thermalin Diabetes, LLC has an exclusive license to ChaB24- related IP, which is owned by CWRU. We thank the Study Section for its guidance.